title: Proteolytic processing and Ca2+-binding activity of dense-core vesicle polypeptides in Tetrahymena.
abstract: Formation and discharge of dense-core secretory vesicles depend on controlled rearrangement of the core proteins during their assembly and dispersal. The ciliate Tetrahymena thermophila offers a simple system in which the mechanisms may be studied. Here we show that most of the core consists of a set of polypeptides derived proteolytically from five precursors. These share little overall amino acid identity but are nonetheless predicted to have structural similarity. In addition, sites of proteolytic processing are notably conserved and suggest that specific endoproteases as well as carboxypeptidase are involved in core maturation. In vitro binding studies and sequence analysis suggest that the polypeptides bind calcium in vivo. Core assembly and postexocytic dispersal are compartment-specific events. Two likely regulatory factors are proteolytic processing and exposure to calcium. We asked whether these might directly influence the conformations of core proteins. Results using an in vitro chymotrypsin accessibility assay suggest that these factors can induce sequential structural rearrangements. Such progressive changes in polypeptide folding may underlie the mechanisms of assembly and of rapid postexocytic release. The parallels between dense-core vesicles in different systems suggest that similar mechanisms are widespread in this class of organelles.
authors.affiliation: Department of Molecular Genetics & Cell Biology, The University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Verbsky
authors.forename: A P
authors.initials: AP
authors.lastname: Turkewitz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002268:Carboxypeptidases / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002918:Chymotrypsin / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D016808:Tetrahymena thermophila / Q000166:cytology*
chemical_list: D002135:Calcium-Binding Proteins; D002614:Chelating Agents; C103333:GRL1 protein, Tetrahymena thermophila; D011498:Protein Precursors; D015800:Protozoan Proteins; D004533:Egtazic Acid; D002268:Carboxypeptidases; D010450:Endopeptidases; D002918:Chymotrypsin; D002118:Calcium